Novel cell penetrating peptide

ABSTRACT

According to the present invention, it is possible to provide a novel cell penetrating peptide that transports proteins into cells and/or into nuclei at higher frequency than conventional cell penetrating peptides, and a pharmaceutical containing the peptide.

TECHNICAL FIELD

The present invention relates to a novel cell penetrating peptide and apharmaceutical containing the peptide.

BACKGROUND TECHNOLOGY

While general proteins, nucleic acids in therapeutic or diagnostic usedo not penetrate the cell membrane, it has recently been found thatthere are peptides that transport proteins, nucleic acids into cells andnuclei in living organisms (hereinafter also referred to as “TATproteins”) (non-patent document 1 to non-patent document 3).Furthermore, it has been found that a fusion protein of a peptideconsisting of 11 particular amino acids of the TAT proteins with anotherprotein penetrates the cell membrane; such regions essential for cellmembrane passage, i.e., cell penetrating peptides, are called PTDs(Protein Transduction Domains) (non-patent document 4).

To date, methods have been developed for transporting proteins, nucleicacids into cells by means of various cell penetrating peptides.Specifically, a method utilizing a particular partial polypeptide of theHIV-1 TAT protein (patent document 1) has been proposed.

However, cell penetrating peptides that have conventionally been usedexist naturally, posing the problem of low translocation efficiency. Forthis reason, there has been a demand for the development of a peptidehaving a high transportation efficiency.

-   Patent document 1: JP-A-HEI-10-33186-   Non-patent document 1: Green, M. et al. Cell 55, 1179-1188 (1988)-   Non-patent document 2: Frankel, A. D. et al. Cell 55, 1189-1193    (1988)-   Non-patent document 3: Fawell, S. et al. Proc. Natl. Acad. Sci. 91,    664-668 (1994)-   Non-patent document 4: Nagahra, H. et al. Nature Medicine 4,    1449-1452 (1998)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

It is an object of the present invention to provide a novel cellpenetrating peptide and a pharmaceutical containing the peptide.

Means for Solving the Problems

The present inventors diligently investigated to provide a peptideconsisting of a novel amino acid sequence, useful as a cell penetratingpeptide, identified the amino acid sequence of a peptide that transportsproteins into cells at higher frequency than conventional cellpenetrating peptides, and developed the present invention. Specifically,the present inventors discovered a peptide with high intracellulartransferability, in a random peptide library of 10¹² molecules bycomprehensive screening. Peptides selected using this technique arethought to be particularly highly transferable among the peptides withthe capability of intracellular translocation.

Accordingly, the present invention is as follows:

(1) A peptide having the amino acid sequence shown below.

B-X-Z-X-Arg-Z-Tyr-J-X-O¹-X-Arg-O²-X-X or X-X-O¹-Arg-X-O²-X-J-Tyr-Z-Arg-X-Z-X-Bwherein (i) B is arginine or lysine, (ii) at least one of O¹ or O² isarginine, (iii) Z is a hydrophobic amino acid, (iv) J is serine oralanine, and (v) X is an arbitrarily chosen amino acid.(2) The peptide according to (1), consisting of the amino acid sequenceshown by any one of SEQ ID NOS:1 to 34 in the sequence listing whereinone or a plurality of amino acids have been substituted, deleted, addedor inserted.(3) A peptide consisting of the amino acid sequence shown by any one ofSEQ ID NOS:35 to 47 in the sequence listing wherein one or a pluralityof amino acids have been substituted, deleted, added or inserted, andthe reversed-chain peptide thereof.(4) A peptide consisting of the amino acid sequence shown by any one ofSEQ ID NOS:1 to 47 in the sequence listing, and the reversed-chainpeptide thereof.(5) A DNA consisting of the DNA sequence that encodes the peptideaccording to any one of (1) to (4) above.(6) A recombinant vector containing the DNA according to (5) above.(7) A transformant comprising the recombinant vector according to (6)above.(8) A peptide-bound substance containing the peptide according to anyone of (1) to (4) above and a bioactive substance.(9) The peptide-bound substance according to (8) above, wherein thebioactive substance is a protein having a bioactivity, a polypeptidehaving a bioactivity, a drug-encapsulated liposome, apolyethylene-glycolated drug-encapsulated liposome, a low-molecularcompound, a nucleic acid, a magnetic bead, a nano-gauge particle or aphage.(10) The peptide-bound substance according to (8) above, wherein theprotein having a bioactivity is an about 10 KDa to about 120 KDa proteinor 4 to 30 polypeptides.(11) The peptide-bound substance according to (8) above, wherein theprotein having a bioactivity is mi-transcription factor (MITF).(12) The peptide-bound substance according to any one of (8) to (11)above, which is transported into cells and/or into nuclei.(13) The peptide-bound substance according to any one of (8) to (11)above, which is transported into cells.(14) A pharmaceutical containing the peptide-bound substance accordingto (8) above.(15) The pharmaceutical according to (14) above, which is used as ananti-allergic drug.

Effect of the Invention

According to the present invention, it is possible to provide a cellpenetrating peptide that transports proteins into cells and/or intonuclei at a higher frequency than conventional cell penetratingpeptides, and a pharmaceutical containing the peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A representation showing results of an intracellulartranslocation test of a fluorescence-labeled peptide. The upper panelsshow fluorescence from FITC; the lower panels show differentialinterference microscopic images of cells in combination withfluorescence images.

FIG. 2 A representation showing results of an intracellulartranslocation test of a fluorescence-labeled peptide using a slidechamber. The upper panels of photographs show fluorescence from FITC;the lower panels show differential interference microscopic images ofcells in combination with fluorescence images.

FIG. 3 A representation showing results of analyses by flowcytometry.

FIG. 4 A representation showing results of intracellular translocationtests of eGFP fusion proteins using a confocal microscope.

FIG. 5 A representation showing the wheel structures of the peptidesKSH1 and KSH1-1 to 1-9 (SEQ ID NO: 33 and SEQ ID NOS: 1-9,respectively). In the drawings below, abbreviations for amino acids usedin wheel structures are defined as follows: A: alanine, V: valine, L:leucine, I: isoleucine, P: proline, F: phenylalanine, W: tryptophan, M:methionine, G: glycine, S: serine, T: threonine, C: cysteine, Y:tyrosine, N: asparagine, Q: glutamine, E: glutamic acid, K: lysine, R:arginine, H: histidine, D: aspartic acid.

FIG. 6 A representation showing results of analyses of thefluorescence-labeled peptides KSH1 (SEQ ID NO: 33) and KSH1-1 to 1-9(SEQ ID NOS: 1-9) in CHO cells by flow cytometry (FACS).

FIG. 7 A representation showing the wheel structures of the peptidesKSH1-11 to 1-24 (SEQ ID NOS: 10-23) and KSH1-35 (SEQ ID NO: 32).

FIG. 8 A representation showing results of analyses of thefluorescence-labeled peptides KSH1-11 to 1-24 (SEQ ID NOS: 10-23) andKSH1-35 (SEQ ID NO: 32) in CHO cells by flowcytometry.

FIG. 9 A representation showing the wheel structures of the peptidesKSH1-24 to 1-26 (SEQ ID NOS: 23-25) and KSH1-28 to 1-30 (SEQ ID NOS:27-29).

FIG. 10 A representation showing results of analyses of thefluorescence-labeled peptides KSH1-1, KSH1-24 to 1-26 (SEQ ID NOS:23-25) and KSH1-28 to 1-30 (SEQ ID NOS: 27-29) in CHO cells byflowcytometry.

FIG. 11 A representation showing the wheel structures of the peptidesKSH1-27 (SEQ ID NO: 26), KSH1-33 (SEQ ID NO: 30), KSH1-34 (SEQ ID NO:31) and KSH1-36 (SEQ ID NO: 34).

FIG. 12 A representation showing results of analyses offluorescence-labeled peptides of KSH1-27 (SEQ ID NO: 26), KSH1-33 (SEQID NO: 30), KSH1-34 (SEQ ID NO: 31) and KSH1-36 (SEQ ID NO: 34) in CHOcells by flowcytometry.

FIG. 13 A representation showing the wheel structure of a peptide of anamino acid sequence obtained from results of an investigation of amodification of the peptide KSH1.

FIG. 14 A representation showing construct of the expression of eGFPfusion proteins. The depicted linker sequences GGGS and GGGSS arerespectively described by SEQ ID NOS: 120 and 121.

FIG. 15 Vector maps of eGFP fusion proteins.

FIG. 16 A representation showing how to insert synthetic oligos into avector. This drawing is for a type D vector.

FIG. 17 A representation showing results of intracellular translocationtests of eGFP fusion proteins using a confocal microscope. The upperpanels show fluorescence from FITC; the lower panels show differentialinterference microscopic images of cells in merged with fluorescenceimages.

FIG. 18 A representation showing the wheel structures of the peptidesKSH2 to KSH10 (SEQ ID NO: 43 and SEQ ID NOS: 35-42, respectively).

FIG. 19 A representation showing results of intracellular translocationtests with the addition of 25 μM of each fluorescence-labeled peptide.The upper panels show fluorescence from FITC; the lower panels showdifferential interference microscopic images of cells in combinationwith fluorescence images.

FIG. 20 A representation showing the wheel structures of the peptidesKSH2 (SEQ ID NO: 43) and KSH2-1 to 2-4 (SEQ ID NOS: 44-47).

FIG. 21 A representation showing results of intracellular translocationtests with the addition of 50 μM, 25 μM and 12.5 μM of eachfluorescence-labeled peptide. The upper panels show fluorescence fromFITC; the lower panels show differential interference microscopic imagesof cells in combination with fluorescence images.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is hereinafter described in detail.

(1) Peptides

In the present invention, peptides include the L-form or D-form of apeptide having the amino acid sequenceB-X-Z-X-Arg-Z-Tyr-J-X-O¹-X-Arg-O²-X-X orX-X-O¹-Arg-X-O²-X-J-Tyr-Z-Arg-X-Z-X-B. In this amino acid sequence, B isarginine or lysine, and at least one of O¹ or O² is arginine, and Z is ahydrophobic amino acid, and J is serine or alanine, and X is anarbitrarily chosen amino acid. Here, B is desirably arginine. If atleast one of O¹ or O² is arginine, the other may be an arbitrarilychosen amino acid. The hydrophobic amino acid for Z is any one ofleucine, phenylalanine, isoleucine, valine, tyrosine, and tryptophan,and is desirably leucine, phenylalanine, isoleucine or tryptophan, andis most desirably isoleucine or tryptophan.

Having the amino acid sequence B-X-Z-X-Arg-Z-Tyr-J-X-O¹-X-Arg-O²-X-X orX-X-O¹-Arg-X-O²-X-J-Tyr-Z-Arg-X-Z-X-B may be having an amino acidsequence of the 15 amino acids shown by the above-described sequencesonly, or may be having one or a plurality of arbitrarily chosen aminoacids on the C-terminal side and/or N-terminal side of theabove-described amino acid sequence. Also meant by the same is that thepeptide is capable of transporting proteins into cells and/or intonuclei. The arbitrarily chosen amino acids are not particularly limited,and are desirably basic amino acids (arginine, histidine, lysine),tryptophan, proline, glycine, cysteine and alanine, more desirablyglycine, cysteine and arginine. The number thereof is not particularlylimited.

As a more desirable peptide of the peptides shown by the amino acidsequence B-X-Z-X-Arg-Z-Tyr-J-X-O¹-X-Arg-O²-X-X orX-X-O¹-Arg-X-O²-X-J-Tyr-Z-Arg-X-Z-X-B, the L-form or D-form of a peptideconsisting of one of the amino acid sequences shown by SEQ ID NOS:1 to34 in the sequence listing and the reversed-chain peptide thereof can bementioned. As a still more desirable peptide, the L-form or D-form of apeptide consisting of the amino acid sequence shown by SEQ ID NO:1, 2,4, 7, 8, 9, 10, 11, 13, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28, 29, 30,32, 33 or 34 in the sequence listing can be mentioned. The mostdesirable peptide is the amino acid shown by SEQ ID NO:1, 7, 8, 9, 11,13, 17, 18, 19, 20, 22, 25, 26, 27, 29, 30, 32, 33 or 34 in the sequencelisting.

In the present invention, a reversed-chain peptide refers to a peptideobtained by reverting the arrangement of amino acids from the N-terminusto C-terminus of a certain sequence, for example,(N-terminus)-A-B-C-D-(C-terminus), to have the peptide(N-terminus)-D-C-B-A-(C-terminus).

In the present invention, peptides also include, in addition to theaforementioned peptides, peptides having the L-form or D-form of apeptide shown by one of SEQ ID NOS:35 to 47 in the sequence listing andthe reversed-chain peptide thereof. Particularly desirable of thesepeptides are the L-form or D-form of a peptide consisting of the aminoacid sequence shown by SEQ ID NO:35, 36, 38, 39, 42, 43, 45, 46 or 47 inthe sequence listing.

Having the L-form or D-form of a peptide and the reversed-chain peptidethereof means arbitrary having one or a plurality of amino acids on theC-terminal side and/or N-terminal side of the 15 amino acid sequencesshown by one of SEQ ID NOS:35 to 47 in the sequence listing. Also meantby the same is that the peptide is capable of transporting proteins intocells and/or into nuclei. Although the arbitrarily chosen amino acidsare not particularly limited, basic amino acids (arginine, histidine,lysine), tryptophan, proline, glycine, cysteine and alanine aredesirable, and glycine, cysteine and arginine are more desirable. Thenumber thereof is not particularly limited.

Furthermore, the peptides of the present invention shown by SEQ ID NOS:1to 47 in the sequence listing include peptides having an amino acidsequence wherein 1 or 2 or more amino acids have been deleted, added,inserted or substituted by other amino acids, or peptides having anamino acid sequence consisting of a combination thereof. Also meant bythe same is that the peptide has the capability of translocation tocells. In this case, even if an amino acid has been inserted, deleted orsubstituted, amino acid sequences capable of transporting proteins intocells and/or into nuclei at a frequency similar to that for the peptidesshown by SEQ ID NOS:1 to 47 in the sequence listing can be mentioned. Ifan amino acid has been inserted, deleted or substituted, the position ofthe insertion, deletion or substitution is not particularly limited.

The present invention particularly includes a peptide having threeC-terminal amino acids deleted from the peptide shown by the sequenceB-X-Z-X-Arg-Z-Tyr-J-X-O¹-X-Arg-O²-X-X. The peptide may be a peptidehaving 1 or 2 C-terminal amino acids deleted.

Here, the peptides consisting of the amino acid sequences shown by SEQID NOS:1 to 47 in the sequence listing are novel sequences withouthomology to any known PTDs that have been reported to date, andexhibited no homology to any human cDNA sequences that have beenreported to date.

A peptide of the present invention can be prepared according to acommonly known method of peptide synthesis, and substitution, additionor deletion can easily be achieved by changing the kind of protectedamino acid. Special amino acids such as D-amino acid and sarcosine(N-methylglycine) may be introduced. Methods of peptide synthesisinclude, for example, solid phase synthesis, liquid phase synthesis;after the synthetic reaction, a peptide used in the present inventioncan be purified and isolated by combining ordinary methods ofpurification, for example, solvent extraction, distillation, columnchromatography, liquid chromatography or recrystallization.

Commonly known methods of peptide synthesis include, for example,methods described in (i) to (v) below.

-   (i) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience    Publishers, New York, (1966)-   (ii) Schroeder and Luebke, The Peptide, Academic Press, New York,    1965-   (iii) Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken,    published by Maruzen Co. (1975)-   (iv) Haruaki Yajima and Shunpei Sakakibara: Seikagaku Jikken Koza 1,    Tanpakushitsu no Kagaku IV, 205 (1977)-   (v) Haruaki Yajima, ed.: Zoku Iyakuhin no Kaihatsu, Vol. 14, Peptide    Synthesis, Hirokawa Shoten

(2) DNA Sequences and DNAs

In the present invention, DNAs include a DNA consisting of the DNAsequence that encodes the peptide represented by the amino acid sequenceshown by one of SEQ ID NOS:1 to 47 in the sequence listing;specifically, such DNAs include, but are not limited to, a DNAconsisting of the DNA sequence shown by SEQ ID NO:48 or 49 in thesequence listing. The DNA sequence shown by SEQ ID NO:48 or 49 in thesequence listing encodes the amino acid sequence shown by SEQ ID NO:33or 34 in the sequence listing, respectively.

In the present invention, the DNA contains a DNA consisting of a DNAsequence that hybridizes with a complementary strand of the DNAconsisting of the DNA sequence that encodes the peptide shown by one ofSEQ ID NOS:1 to 47 in the sequence listing under stringent conditions.

In the present invention, the DNA sequence that hybridizes understringent conditions contains a DNA sequence having a homology of about80% or more, preferably about 90% or more, more preferably about 95% ormore, to a complementary strand of the DNA sequence that encodes thepeptide shown by SEQ ID NO:1 or 2 in the sequence listing. Hybridizationcan be performed according to a commonly known method or a method basedthereon, for example, a method described in Molecular Cloning, 2ndedition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) andthe like. High-stringent conditions refer to, for example, conditionsinvolving a sodium concentration of about 19 mM to about 40 mM,preferably about 19 mM to about 20 mM, and a temperature of about 50° C.to about 70° C., preferably about 60° C. to about 65° C. In particular,a case wherein the sodium concentration is about 19 mM and thetemperature is about 65° C. is the most preferable.

The DNA sequences of the present invention include sequences having anidentity of about 50% or more to the DNA sequences that encode thepeptides shown by SEQ ID NOS:1 to 47 in the sequence listing. The DNAsequences of the present invention contain a DNA sequence having anidentity of preferably about 60% or more, more preferably about 70% ormore, still more preferably about 80% or more, still yet more preferablyabout 90% or more, even more preferably about 95% or more.

The DNA sequences of the present invention include the DNA sequencesthat encode the peptides shown by SEQ ID NOS:1 to 47 in the sequencelisting wherein bases have been inserted, deleted or substituted. Here,as the number of bases inserted, deleted or substituted, 1 base or 2bases or more can be mentioned; for example, 1 base to 10 bases,preferably 1 base to 5 bases, can be mentioned. In this case, even if abase has been inserted, deleted or substituted, a DNA sequence capableof transporting proteins into cells and/or into nuclei at a frequencysimilar to that for the DNA sequences that encode the peptides shown bySEQ ID NOS:1 to 47 in the sequence listing can be mentioned. If a basehas been inserted, deleted or substituted, the position of theinsertion, deletion or substitution is not particularly limited.

A DNA of the present invention can be synthesized according to acommonly known method. A cDNA that encodes a fusion protein, ifprepared, can be obtained by amplification by a PCR method usingprimers.

Primers that can be used in the present invention include, for example,a primer for use in preparing a cDNA that encodes a fusion protein witha green fluorescent protein eGFP (Enhanced Green Fluorescent Protein).

(3) Recombinant Vectors and Transformants

Recombinant vectors used in the present invention include vectors thatcan be expressed in prokaryotic cells such as of Escherichia coli (forexample, pBR322, pUC119 or derivatives thereof). Furthermore, ineukaryotic cells, expression vectors is for yeast include, for example,plasmid vectors such as pAUR112 (Takara Bio Inc.). Vectors that can beexpressed in cells derived from mammals include, for example, plasmidvectors like pcDNA3.1 (Invitrogen) and viral vectors such as pDON-AI DNA(Takara Bio Inc.).

A recombinant vector of the present invention are prepared byrecombining the entire or a portion of a DNA having the DNA sequence ofthe present invention with these recombinant vectors by a commonly knownmethod.

A recombinant vector can be obtained by, for example, a method describedin Molecular Cloning, 2nd (J. Sambrook et al., Cold Spring Harbor Lab.Press, 1989).

A transformant of the present invention refers to a transformantcomprising a recombinant vector of the present invention. As the host,Escherichia coli, yeast, animal cells and the like can be used.Escherichia coli is preferable. Auxotrophs and antibiotic-sensitivestrains can also be used as hosts.

A transformant of the present invention can be prepared according to acommonly known method. For example, transformation can be performedaccording to methods described in Molecular Cloning, 2nd (J. Sambrook etal., Cold Spring Harbor Lab. Press, 1989), such as the protoplastpolyethylene glycol method and electroporation method.

(4) Peptide-Bound Substances

In the present invention, peptide-bound substances include onecontaining the peptide shown by the amino acid sequence:B-X-Z-X-Arg-Z-Tyr-J-X-O¹-X-Arg-O²-X-X orX-X-O¹-Arg-X-O²-X-J-Tyr-Z-Arg-X-Z-X-B or the peptide consisting of theamino acid sequence shown by one of SEQ ID NOS:1 to 47 in the sequencelisting and a bioactive substance.

In the present invention, the peptide shown by the amino acid sequence:B-X-Z-X-Arg-Z-Tyr-J-X-O¹-X-Arg-O²—X-X orX-X-O¹-Arg-X-O²-X-J-Tyr-Z-Arg-X-Z-X-B or the peptide shown by one of SEQID NOS:1 to 47 in the sequence listing can be utilized as a cellpenetrating peptide; by binding a peptide of the present invention to abioactive substance, the bioactive substance can be transported intocells and/or into nuclei. More preferably, a bioactive substance istransported into cells. Here, the cells are animal cells, particularlyhuman cells. The human cells may be adhesive cells or floating cells,and may be cells that configure various organs in living organisms.

In the present invention, bioactive substances include proteins having abioactivity, peptides having a bioactivity, drug-encapsulated liposomes,polyethylene-glycolated drug-encapsulated liposomes (hereinafter alsoreferred to as “PEGylated drug-encapsulated liposomes”), low-molecularcompounds, nucleic acids, magnetic beads, nano-gauge particles andphages.

Here, proteins having a bioactivity include proteins for the treatment,prevention and/or diagnosis of disease and the like, being about 10 KDato about 500 KDa proteins; about 10 KDa to about 120 KDa proteins areparticularly desirable. Specifically, such proteins include, but are notlimited to, enzymes, antibodies, transcription factors and partialpeptides thereof. Enzymes include SOD (Molecules and Cells 2005, 19,191-197, W. S. Eum et al.). Antibodies include antibodies againstintracellular proteins, single-chain antibody, antibodies againstforeign proteins such as viruses and the like (Current MolecularMedicine 2004, 4, 519-528, M. N. Lobato and T. H. Rabbitts, MolecularTherapy 2003, 8, 355-366, Y. Y. Wheeler et al. and the like).Transcription factors include mi-transcription factor(microphthalmia-associated transcription factor: hereinafter alsoreferred to as “MITF”) and the like. Here, MITF is a kind oftranscription regulatory factor existing in living organisms, being aprotein capable of regulating c-kit gene expression, characteristic ofmast cells. Specifically, such transcription factors include, but arenot limited to, the various MITFs described in JP-A-2004-201547.

In the present invention, polypeptides having a bioactivity includepeptides for the treatment, prevention and/or diagnosis of disease andthe like, being peptides having 2 to 100 amino acids; peptides having 4to 30 amino acids are particularly desirable. Specifically, suchpeptides include, but are not limited to, HSP (Heat Shock Protein) 20analogue peptide (J Appl Physiol 98: 1836-1845, 2005), the KLAKantibacterial peptide (Cancer Research 61, 7709-7712, 2001), HIF-1α(Proc Natl Acad Sci USA 99: 10423-10428, 2002), PKC (Protein Kinase C) δinhibitory peptide (Proc Natl Acad Sci USA 98: 11114-11119, 2001), VIVIT(Nature Medicine 10: 305-309, 2004) and the like.

In the present invention, liposomes include small unilamellar vesicles(hereinafter also referred to as “SUV”), large unilamellar vesicles(hereinafter also referred to as “LUV”), multilamellar vesicles(hereinafter also referred to as “MLV”) and the like; SUV or LUV ispreferable. Furthermore, drug-encapsulated liposomes includeanti-inflammatory drugs such as diclofenac sodium and tobramycinencapsulated in liposomes described above.

In the present invention, PEGylated drug-encapsulated liposomes includea drug-encapsulated liposome having polyethylene glycol (PEG) bound tothe surface thereof.

A drug-encapsulated liposome or PEGylated drug-encapsulated liposome inthe present invention can be prepared by methods described inJP-A-HEI-4-346918, JP-A-HEI-10-29930, the pamphlet for InternationalPatent Application Publication No. 97/29128 or the pamphlet forInternational Patent Application Publication No. 01/064743.

In the present invention, low-molecular compounds include, for example,anti-inflammatory drugs such as diclofenac sodium, tobramycin andcyclosporine.

In the present invention, nucleic acids include, for example, plasmids,siRNAs and antisense DNA for disease-related genes.

In the present invention, magnetic beads include, for example,supermagnetic ion oxide particles introduced to T cells, B cells, ormacrophage for tracking the localization of such cells by MRI (AdvancedDrug Delivery Reviews 57: 637-651, 2005).

In the present invention, nano-gauge particles include, for example,nano-sized particles having proteins, low-molecular compounds, nucleicacids, polysaccharides and the like encapsulated therein (Advanced DrugDelivery Reviews 57: 637-651, 2005).

In the present invention, phages include, for example, M13 phagesincorporating various cDNA expression units (Advanced Drug DeliveryReviews 57: 529-546, 2005).

In the present invention, a peptide-bound substance containing a peptideof the present invention and a protein having a bioactivity as describedabove can be obtained by culturing host cells transformed using arecombinant vector comprising a DNA that encodes the substance, andisolating the protein produced thereby by a method such as highperformance liquid chromatography (HPLC). The DNA used here may be a DNAcomprising a fusion of the gene that encodes the peptide of the presentinvention and the gene that encodes a protein or polypeptide having abioactivity as described above. Alternatively, to obtain a peptide-boundsubstance containing a peptide of the present invention and a proteinhaving a bioactivity, the respective genes may be expressed to acquirethe peptide of the present invention and the protein or polypeptidehaving a bioactivity, and they may be fused by a chemical reaction. As ameans of the fusion by a chemical reaction, disulfide linkage using acysteine residue and the like can be mentioned. Alternatively, apeptide-bound substance containing a peptide of the present inventionand a protein having a bioactivity can be prepared by a method using achemical crosslinking agent. In this case, it is preferable to preventcrosslinking between the peptide of the present invention and thefunctional active site of the protein having a bioactivity; as themethod of chemical crosslinking, a method described in JP-A-HEI-10-33186and the like can be mentioned.

Furthermore, by using a method of chemical crosslinking, a peptide ofthe present invention can also be bound to a drug-encapsulated liposome,a PEGylated drug-encapsulated liposome or a low-molecular compound.

By binding a peptide in the present invention to a drug-encapsulatedliposome or a PEGylated drug-encapsulated liposome, the drugencapsulated in the liposome can be delivered into cells. Methods ofpeptide binding in the present invention include, for example, a methodwherein a cysteine residue is introduced to the N-terminus orC-terminus, and binding the peptide to a drug-encapsulated liposomehaving a maleimide group or a PEGylated drug-encapsulated liposome viaan SH group and the like.

(5) Pharmaceuticals

A peptide-bound substance of the present invention can be utilized as apharmaceutical. Furthermore, a desired bioactive substance can betransported into cells and/or into nuclei by binding a peptide in thepresent invention to the bioactive substance; therefore, depending onthe kind of the bioactive substance to be bound, the peptide of thepresent invention can be utilized as a therapeutic drug and/orprophylactic drug for a wide variety of diseases. The bioactivesubstance to be bound to the peptide of the present invention ispreferably a protein having a bioactivity. More preferably, by binding apeptide of the present invention to an MITF mutant, the peptide of thepresent invention can be utilized as an anti-allergic drug.

If used as a pharmaceutical, a peptide-bound substance in the presentinvention can be prepared as a preparation and administered according toa commonly known method. For example, the peptide-bound substance can beadministered to humans or other mammals in the form of a liquid as is,or as a pharmaceutical composition in the form an appropriate dosageform, orally or parenterally.

For producing a solution and the like, an appropriate solvent orsuspending agent can be used.

In producing tablets or capsules as an appropriate dosage form, anappropriate excipient can be used. Liquid preparations for oraladministration, i.e., syrups, suspensions, solutions and the like,comprise a commonly used inert diluent. The preparations can alsocomprise, in addition to the inert diluent, auxiliaries, for example,wetting agents, suspension aids, sweetening agents, flavoring agents,coloring agents, preservatives, stabilizers and the like.

The dosage of a peptide-bound substance in the present invention forhumans is determined according to the age, body weight, general healthstatus, sex, diets, duration of administration, method ofadministration, excretion rate, combination of drugs, and the severityof the patient's condition being treated, in consideration of these andother factors. For example, the dosage is about 0.01 mg/Kg to about 1.0mg/Kg, and can be administered once daily or in divided portions.

EXAMPLES

The present invention is hereinafter described specifically on the basisof the following examples, to which, however, the present invention isnever limited.

Example 1 Cell Membrane Passage Test Using Synthetic Peptides Example1-1 Section of Cell Penetrating Peptide Sequences

Intracellularly transferable peptide sequences were concentratedaccording to a method described in JP-A-2005-13073, using a library ofJurkat cells (ATCC NO. TIB-152). Specifically, after a library of invitro viruses presenting random peptides of 15 amino acids (hereinafterabbreviated IVVs) (library scale 10¹²) was prepared, the library wasadded to HeLa cells (ATCC NO. CCL2) or Jurkat cells. After cDNAs of IVVsthat had translocated into the cells were recovered by PCR, IVVs wereagain prepared and added to the cells. By repeating theaddition-recovery operation, “the peptides that translocation intocells” being present in the library were concentrated. To identify thelibraries wherein the intracellularly transferable peptides had beenconcentrated in the various concentration operation stages, 11 kinds ofamino acid sequences were arbitrary selected from the libraries afterthe 5th to 8th concentration operations, and the peptides were examinedfor the capability of intracellular translocation.

Example 1-2 Confocal Microscopic Analysis

With the PTD derived from HIV TAT protein shown by SEQ ID NO:50 in thesequence listing as the positive control (in FIG. 1 to FIG. 3, denotedby “Positive” or “TAT”), and a mutant of the TAT-derived PTD shown bySEQ ID NO:51 in the sequence listing, reported by Ulo Langel et al.(Cell-Penetrating Peptides: Processes and Applications, Series:Pharmacology and Toxicology: Basic and Clinical Aspects Volume:3, 2002),as the negative control (in FIG. 1, denoted by “Negative”), the peptidesof the 11 sequences selected in Example 1-1 were evaluated.

The peptides of the 11 sequences selected in Example 1-1 weresynthesized by the solid phase method, and fluorescently labeled bycoupling 5,6-Carboxyfluorescein to the N-terminus of each peptide.Furthermore, the peptides were purified by HPLC to obtain a purity of70% or more. Subsequently, each fluorescently labeled peptide wasdissolved in 10% Dimethyl Sulfoxide (DMSO) to obtain 1 mM (hereinafterreferred to as peptide solution).

Three kinds of cells were used for incorporating the peptides: CHO cells(ATCC NO. CCL-61), HeLa cells, and Jurkat cells. 10⁴ CHO cells and HeLacells and 10⁵ Jurkat cells were separately inoculated to a 96-wellplate, and cultured for 2 or 3 days. Subsequently, when the cells becameconfluent, each peptide solution was added to each type of cell, and thecells were incubated for 1 hour. After each type of cell was washed withPBS three times, 100 μL of 0.25% trypsin-1 mM EDTA solution was added,and trypsinization was performed at room temperature for 5 minutes.After 400 μL of PBS (10% FCS) was added to neutralize the trypsin, thecells were washed with 1 mL of HBSS (Hanks' Balanced SaltSolution/produced by Invitrogen) three times. After the washing, thecells were suspended in 50 μL of HBSS and analyzed using a confocalmicroscope. Measurements and analysis were performed using a confocaldifferential interference laser microscope system (Bio-Rad,Radience2100/Green He—Ne, 488 nm, microscope: Nikon, ECLIPSE E600).

As a result, the peptide of the novel PTD candidate sequence shown bySEQ ID NO:33 in the sequence listing (in FIG. 1 to FIG. 3, denoted by“KSH1”) exhibited the highest intracellular transferability. The resultsfor the KSH1 peptide, positive control and negative control are shown inFIG. 1. In all of the CHO cells, HeLa cells and Jurkat cells,fluorescence from the KSH1 peptide was observed. The fluorescenceintensity from the KSH1 peptide was higher than the fluorescenceintensity of the positive control. Furthermore, the intracellularlytransported peptide was not localized, but detected both in the nucleusand in cytoplasm.

From these results, the KSH1 peptide was found to translocate intocells. It was also found that the amount thereof exceeded that of theHIV-derived TAT peptide, a conventionally known PTD.

Furthermore, CHO cells were inoculated to a slide chamber (produced byNunc), 25 μM of each of the KSH1 peptide and positive control was addedto the CHO cells, and the intracellular transferability thereof wasdetermined using a confocal microscope.

The results are shown in FIG. 2.

From these results, it was found that the KSH1 peptide was translocatedinto all CHO cells.

Example 1-3 Flowcytometry Analysis

Each peptide solution, adjusted to 1 mM, was diluted with a medium tothree levels, 100 μM, 50 μM, and 25 μM, and added to cells. Three kindsof cells were used: CHO cells, HeLa cells, and Jurkat cells. After eachpeptide solution was added, the cells were incubated for 1 hour. Afterthe cells were washed with PBS three times, 100 μL of 0.25% trypsin-1 mMEDTA solution was added, and trypsinization was performed at roomtemperature for 5 minutes. After 400 μL of PBS (10% FCS) was added tostop the trypsinization, the cells were washed with 1 mL of HBSS threetimes. After the washing, the cells were suspended in 500 μL of PBS (10%FCS) and subjected to flowcytometry (hereinafter, FACS) analysis usingFACS Calibur (Becton Dickinson).

The results are shown in FIG. 3. The ordinate indicates cell number; theabscissa indicates fluorescence intensity.

From these results, it was found that the KSH1 peptide exhibited 3 to 10times higher intracellular transferability than the positive control,having a potent capability of translocation.

Example 2 Confirmation of Translocation Activity Using eGFP FusionProtein Example 2-1 Construction of His-eGFP Expression Vector

With an eGFP cDNA (eGFP-NII) (produced by Amersham Pharmacia) as thetemplate, “a His-eGFP expression vector” having a His-Tag andrestriction endonuclease recognition sites was constructed by a PCRmethod.

PCR was performed using the Easy-A PCR kit (produced by Stratagene). AneGFP cDNA (eGFP-NII) DNA for the template was diluted to 200 ng/mL.Synthetic DNAs for the primers were adjusted to 20 μM. The template, theFw primer, and the RV primer, each 1 μl, 10 μl of Easy-A PCR buffer, 8μL of dNTPs (2.5 mM), and 0.5 μL of Easy-A were mixed, and this mixturewas filled up with sterile distilled water to make 100 μL. PCRamplification conditions were as follows: 94° C. for 2 minutes, 1 cycle;and 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 2minutes, 30 cycles.

Here, the PCR was performed using the Fw primer shown by SEQ ID NO:116in the sequence listing (Nco-His-GFP-f) and the Rv primer shown by SEQID NO:117 in the sequence listing (GFP-Rev-Bam).

Next, for cloning into a vector, the PCR-amplified fragment was digestedwith the restriction endonucleases NcoI and BamHI, and a 0.8-kb DNAfragment recovered. The recovered DNA fragment was inserted into pET14b.The thus-constructed “His-eGFP expression vector” is a vector whereinthe His-eGFP fusion protein is expressed under the control of the T7promoter.

Example 2-2 Construction of His-Novel PTD Candidate Sequence-eGFPExpression Vector

By a PCR method, with an eGFP cDNA (eGFP-NII) as the template, “aHis-KHS1-eGFP expression vector” having a His-Tag, a novel PTD candidatesequence and restriction endonuclease recognition sites was constructed.

PCR was performed using the Fw primer shown by SEQ ID NO:118 in thesequence listing (KSH-kp-nd-GF-f) and the Rv primer shown by SEQ IDNO:117 in the sequence listing (GFP-Rev-Bam). Further PCR was performedwith the amplified fragment as the template, using the Fw primer shownby SEQ ID NO:119 in the sequence listing (KSH-kp-nd-f2) and the Rvprimer shown by SEQ ID NO:117 in the sequence listing (GFP-Rev-Bam).

Subsequently, for cloning into a vector, the PCR-amplified fragment wasdigested with the restriction endonucleases NcoI and BamHI, and a 0.8-kbDNA fragment was recovered. The recovered DNA fragment was inserted intopET14b.

The thus-constructed “His-KHS1-eGFP expression vector” has theHis-KSH1-eGFP fusion protein expressed under the control of the T7promoter.

Example 2-3 Preparation of eGFP Fusion Proteins

After the BL21LysS strain, derived from Escherichia coli strain B, wastransformed with the “His-eGFP expression vector” and the “His-KSH1-eGFPexpression vector” constructed in Examples 2-1 and 2-2, respectively,each transformant was precultured in 20 mL of LB (37° C., 15 hours).

The preculture broth was inoculated to 1 L of LB at 2%, and cultured at37° C. for 2.5 hours. IPTG was added at a final concentration of 1 mM,and further cultivation was performed for 4 hours. After centrifugation(4000 rpm, 20 min Hitachi himac CR7) and bacterial cell collection, thebacterial cells were suspended in 50 mL of PBS. Furthermore, thesuspension was centrifuged (3500 rpm, 20 min KUBOTA 5200), and bacterialcells were collected. Subsequently, the bacterial cells were suspendedin 30 mL of PBS and freeze-thawed three times. After addition of 15 μLof DNase solution (Benzonase Takara Bio NV677) and bacterial cellincubation at room temperature for 10 minutes, centrifugation wasperformed (18000 rpm, 20 min TOMY UD-201), and the supernatant wasrecovered. The supernatant was applied to a PBS-equilibrated Ni-NTAcolumn (QIAGEN 30430). After the column was washed with 50 mL of a PBScontaining 10 mM imidazole, the fusion proteins were eluted with 4 mL ofa PBS containing 200 mM imidazole. 15 μL of the eluate waselectrophoresed by SDS-PAGE (PAG-Mini, 4-20% gradient gel, Daiichi PureChemicals), the gel after electrophoresed was stained using Quick CBB(Wako Pure Chemical Industries, 299-50101), and detection of proteinswas attempted.

As a result, bands with desired sizes corresponding to the His-KSH1-eGFPfusion protein and the His-eGFP fusion protein were detected.

Example 2-4 Confirmation of Intracellular Translocation of KSH1 FusionProtein

An intracellular translocation test of each fusion protein was performedusing CHO cells.

CHO cells or HeLa cells were inoculated to a 96-well plate at 10⁴ cellsper well; 2 days later, the cells were used after becoming confluent.After the cells were washed with MEM medium three times, a peptidesolution comprising the His-novel PTD candidate sequence-eGFP fusionprotein and a peptide solution comprising the His-eGFP fusion protein,each 100 μL, was added, and the cells were incubated at 37° C. for 1hour. After the cells were washed with PBS three times, the cells wererecovered via trypsinization. After the recovered cells were washed withMEM medium one time, and with PBS (10% FCS) three times, the cells weresuspended in 100 μL of PBS (10% FCS) and subjected to confocalmicroscopy.

As a result of the confocal microscopy, fluorescence from eGFP wasdetected in the cells to which the peptide solution comprising theHis-novel PTD candidate sequence-eGFP fusion protein had been added.

The results are shown in FIG. 4.

From these results, it was found that the His-novel PTD candidatesequence-eGFP fusion protein (in FIG. 4, denoted by “KSH1”) wastranslocated into the cells. Meanwhile, no translocation of His-eGFP (inFIG. 4, denoted by “His”) into the cells was detected.

Furthermore, because the His-novel PTD candidate sequence-eGFP fusionprotein was detected in a dot-by-dot pattern, its localization inendosome was demonstrated. This disagrees with the fact that the peptidealone is not localized in the cells as shown in Example 1-2. PTD fusionproteins have been reported to be incorporated into cells bymacropinocytosis and localized in endosome (J Control Release. 2005 Jan.20; 102(1):247-53 Cationic TAT peptide transduction domain enters cellsby macropinocytosis. Kaplan I M, Wadia J S, Dowdy S F.). Hence, it wassuggested that the novel PTD candidate sequence might penetrate the cellmembrane by the same mechanism as other PTD peptides.

Example 3 Intracellular Translocation Test Using Synthetic Peptides ofModified PTD Sequences

In the Examples and drawings below, the sequence identification numbersin the sequence listing corresponding to the names of the individualpeptides are as shown in Table 1-1 and Table 1-2.

TABLE 1-1 indication in Examples and Figures SEQ ID NO: KSH1-1 1 KSH1-22 KSH1-3 3 KSH1-4 4 KSH1-5 5 KSH1-6 6 KSH1-7 7 KSH1-8 8 KSH1-9 9 KSH1-1110 KSH1-12 11 KSH1-13 12 KSH1-14 13 KSH1-15 14 KSH1-16 15 KSH1-17 16KSH1-18 17 KSH1-19 18 KSH1-20 19 KSH1-21 20 KSH1-22 21 KSH1-23 22KSH1-24 23 KSH1-25 24 KSH1-26 25 KSH1-27 26

TABLE 1-2 KSH1-28 27 KSH1-29 28 KSH1-30 29 KSH1-33 30 KSH1-34 31 KSH1-3532 KSH1 33 KSH1-36 34 KSH3 35 KSH4 36 KSH5 37 KSH6 38 KSH7 39 KSH8 40KSH9 41 KSH10 42 KSH2 43 KSH2-1 44 KSH2-2 45 KSH2-3 46 KSH2-4 47Positive or TAT 50 Negative 51

Example 3-1 Analysis of KSH1-1 to KSH1-9

Wheel structure prediction (Trends Genet. 16(6): 276-7,2000) of theprimary sequence of the KSH1 peptide (SEQ ID NO:33 in the sequencelisting) revealed characteristic findings of the formation of alysine/arginine cluster, the presence of two tryptophans, thecontainment of proline, a rigid amino acid, and the presence of arginineat a position adjoining to tryptophan, though the arginine was notinvolved in the cluster formation. Hence, in order to determine theimplication of these characteristic sequences in the intracellulartranslocation, SEQ ID NOS:1 to 9 in the sequence listing were designed(FIG. 5). Each peptide was synthesized, fluorescently labeled, purifiedand dissolved in the same manner as Example 1-2, a test of translocationinto CHO cells was performed, and an evaluation was performed byconfocal microscopic analysis and FACS. FACS analysis was performed inthe same manner as Example 1-3. For the KSH1 peptide and modified PTDsequences, i.e., KSH1-1 to KSH1-9 peptides (SEQ ID NOS:1 to 9 in thesequence listing), fluorescence intensities determined by FACS analysisare shown in FIG. 6. The peptide addition concentrations were 50 μM, 25μM, 12.5 μM and 6.5 μM.

When the lysine of the lysine/arginine cluster was substituted witharginine (KSH1-1), at a concentration of 50 μM, the translocationefficiency decreased by about 20% compared with KSH1; however, when thepeptide concentration was reduced, the intracellular translocation wasaccentuated compared with KSH1 (about 2 times at 25 μM, about 8 times at12.5 μM, about 2 times at 6.25 μM). This demonstrated the importance ofthe lysine/arginine cluster.

Next, to examine the role of the arginine not involved in the clusterformation, out of the three arginines being present in KSH1, thearginine was substituted with alanine. When the arginine was substitutedwith alanine, the intracellular translocation decreased remarkably, withintracellular translocation detected only at 50 μl (KSH1-3). It wasfound that this arginine existing in the vicinity of tryptophan plays animportant role in the intracellular translocation.

To examine the significance of proline, which is thought to influencethe conformation of peptides, proline was substituted with alanine; noinfluence on the intracellular transferability was observed (KSH1-2).

Tryptophan is an amino acid characteristic of KSH1, and there are twotryptophans located at the poles of the lysine/arginine cluster on thewheel structure as sandwiching threonine. When the two tryptophans wereboth substituted with alanine, the intracellular translocation was nolonger observable at all (KSH1-6). From this finding, it was found thattryptophan was important to the intracellular translocation.

Whether the intracellular transferability was changed by increasing thenumber of tryptophans was examined. When threonine was substituted withtryptophan to form a tryptophan cluster (KSH1-7), the intracellulartransferability was accentuated remarkably. Compared with KSH1, not lessthan 3 times higher intracellular translocation was detected between 25μM and 6.15 μM, and not less than 10 times higher intracellulartranslocation was detected at 12.5 μM. The accentuation of thetransferability showed the same profile as KSH1-1. Meanwhile, even whenthree tryptophans were uniformly arranged on the wheel structure, theintracellular translocation did not change (KSH1-4). From this finding,it was thought that the intracellular transferability was accentuatedbecause of the formation of the tryptophan cluster.

Since the transferability was accentuated by forming the tryptophancluster, and bearing in mind the results for KSH1-1, lysine wassubstituted with arginine (KSH1-8); the intracellular translocation at50 μM decreased, but the translocation at 25 μM or less was accentuated.Since enlarging the tryptophan cluster was expected to accentuate theintracellular translocation, the number of tryptophans was increased to4 (KSH1-9), but the same tendency as KSH1-8 was obtained.

Example 3-2 Analysis of KSH1-11 to 1-23 and KSH1-35

To determine which amino acids are important to the intracellulartranslocation, comprehensive alanine substitution was performed on thebasis of KSH1-1 to design KSH1-11 (SEQ ID NO:11 in the sequence listing)to KSH1-23 (SEQ ID NO:22 in the sequence listing) and KSH1-35 (SEQ IDNO:32 in the sequence listing) (FIG. 7). Each peptide was synthesized,fluorescently labeled, purified and dissolved in the same manner asExample 1-2, a test of translocation into CHO cells was performed, andan evaluation was performed by confocal microscopic analysis and FACS.FACS analysis was performed in the same manner as Example 1-3. For KSH1and modified PTD sequences, i.e., KSH11-11 to 23 and KSH1-35,fluorescence intensities determined by FACS analysis are shown in FIG.8. The peptide addition concentrations were 50 μM, 25 μM, 12.5 μM and6.5 μM.

When arginine and tryptophan were substituted with alanine, theintracellular translocation decreased clearly. When the 7th tyrosine wassubstituted with alanine, almost no intracellular translocation wasobserved (KSH1-17); it was found that the 7th tyrosine was important tointracellular translocation. Even when the 1st arginine was substitutedwith alanine, the intracellular translocation did not decrease a lot(KSH1-11). When other arginines were substituted with alanine, theintracellular translocation decreased (KSH1-15 and KSH 1-22); however,compared with the 13th arginine substituted (KSH1-3), the reduction inthe intracellular translocation was minor.

Regarding tryptophan, the 6th tryptophan was more important to theintracellular translocation than the 3rd (KSH1-13, KSH1-6). From thesefindings, it was thought that the 13th arginine, the 6th arginine andthe 7th tyrosine played an important role in the intracellulartranslocation, and that other amino acids made a minor contribution tothe intracellular translocation (KSH1-11, KSH1-12, KSH1-14, KSH1-19,KSH1-20, KSH1-23, KSH1-35).

The only case where alanine substitution accentuated the intracellulartranslocation was the substitution of the 8th serine with alanine.

Example 3-3 Analysis of KSH1-24 to 1-26 and KSH1-28 to 1-30

In an attempt to substitute two tryptophans with other hydrophobic aminoacids, KSH11-24 (SEQ ID NO:23 in the sequence listing) to KSH1-26 (SEQID NO:25 in the sequence listing) and KSH1-28 (SEQ ID NO:27 in thesequence listing) to KSH1-30 (SEQ ID NO:29 in the sequence listing) weredesigned (FIG. 9).

Each peptide was synthesized, fluorescently labeled, purified anddissolved in the same manner as Example 1-2, a test of translocationinto CHO cells was performed, and an evaluation was performed byconfocal microscopic analysis and FACS analysis. FACS analysis wasperformed in the same manner as Example 1-3. For KSH1 and modified PTDsequences, i.e., KSH11-24 to 1-26 and KSH1-28 to 1-30, fluorescenceintensities determined by FACS analysis are shown in FIG. 10. Thepeptide addition concentrations were 50 μM, 25 μM, 12.5 μM and 6.5 μM.

First, two tryptophans were substituted with leucine (KSH1-24),phenylalanine (KSH1-25) and isoleucine (KSH1-26); the intracellulartranslocation decreased with leucine, but was nearly the same as KSH1-1with phenylalanine and isoleucine. Hence, with reference to KSH1-18, amodification was designed to substitute the 8th serine of KSH1-26 withalanine, but the intracellular translocation was accentuated verylittle.

Furthermore, two tryptophans were substituted with other hydrophobicamino acids, i.e., valine and tyrosine (KSH1-29, KSH1-30); theintracellular translocation decreased slightly with valine, but goodintracellular translocation was exhibited with tyrosine. Summarizingthese results for hydrophobic amino acids to substitute tryptophan, theintracellular translocation was better in the order of isoleucine,tyrosine, phenylalanine, valine, and leucine.

Example 3-4 Analysis of KSH1-27, KSH1-33, KSH1-34 and KSH1-36

KSH1-27 (SEQ ID NO:26 in the sequence listing), KSH1-33 (SEQ ID NO:30 inthe sequence listing), KSH1-34 (SEQ ID NO:31 in the sequence listing)and KSH1-36 (SEQ ID NO:34 in the sequence listing) were designed (FIG.11).

Each peptide was synthesized, fluorescently labeled, purified anddissolved in the same manner as Example 1-2, a test of translocationinto CHO cells was performed, and an evaluation was performed byconfocal microscopic analysis and FACS analysis. FACS analysis wasperformed in the same manner as Example 1-3. For KSH1 and modified PTDsequences, i.e., KSH1-27, KSH1-33, KSH1-34 and KSH1-36, fluorescenceintensities determined by FACS analysis are shown in FIG. 12. Thepeptide addition concentrations were 50 μM, 25 μM, 12.5 μM and 6.5 μM.

On the basis of the results for KSH1-18, serine and threonine were allsubstituted with alanine (KSH1-27); compared with KSH1-1, theintracellular translocation decreased slightly, but the translocation atlow concentrations was nearly the same.

In KSH1-3, when the arginine (13th) existing in the vicinity of the 6thtryptophan on the wheel structure was substituted with alanine, theintracellular translocation became observable very little. Hence, todetermine which position of arginine is important in KSH1-33, in thevicinity of tryptophan on the wheel structure or at the 13th position,the 13th arginine was substituted with alanine, and the 10th threoninewas substituted with arginine. As a result, the intracellulartranslocation was nearly equivalent or more than KSH1-1 in the sequencelisting. From this finding, it was concluded that the presence ofarginine in the vicinity of the 6th tryptophan on the wheel structurewas important.

Furthermore, to determine whether the 13th arginine is no longernecessary if arginine is present in the vicinity of the 3rd tryptophanin KSH1-34, the 14th tyrosine existing in the vicinity of the 3rdtryptophan on the wheel structure was substituted with arginine. As aresult, the intracellular translocation decreased considerably; it wasconcluded that the presence of arginine in the vicinity of the 6thtryptophan on the wheel structure was essential to the intracellulartranslocation.

KSH1-36, the reversed-chain peptide of KSH1, exhibited accentuatedintracellular translocation at high concentrations compared with KSH1,but the intracellular translocation conversely tended to decrease at lowconcentrations.

Example 3-5 Intracellular Translocation Experiments in HeLa Cells andJurkat Cells

In HeLa cells as with CHO cells, intracellular translocation experimentsof synthetic peptides of modified PTD sequences were performed byconfocal microscopic analysis. Compared with CHO cells, theintracellular translocation was generally lower, but the tendency wasnearly the same as CHO cells. However, in HeLa cells than in CHO cells,the intracellular translocation of KSH1-7 was lower, and conversely theintracellular translocation of KSH1-16 was slightly higher. Inconclusion, better intracellular translocation in HeLa cells wasobserved in the modified peptides KSH1-1, KSH1-8, KSH1-9, KSH1-18,KSH1-20 and KSH1-33.

In Jurkat cells as well, intracellular translocation experiments ofsynthetic peptides of modified PTD sequences were performed by confocalmicroscopic analysis. Compared with CHO cells, the intracellulartranslocation was generally considerably lower, but the tendency wasnearly the same as CHO cells and HeLa cells. However, when tryptophanwas substituted with other hydrophobic amino acids, the intracellulartranslocation decreased considerably. In conclusion, betterintracellular translocation in Jurkat cells was observed in the modifiedpeptides of SEQ ID NOS:KSH1-1, KSH1-7, KSH1-8, KSH1-9, KSH1-12, KSH1-14,KSH1-18, KSH1-20, KSH1-21 and KSH1-33 in the sequence listing, ascompared to that of SEQ ID NO:37 in the sequence listing.

From the results of Examples 3-1 to 3-5, the sequences shown in FIG. 13were identified. In the figure, B is arginine or lysine, either O¹ or O²is arginine, Z is a hydrophobic amino acid, J is serine or alanine, andX is an arbitrarily chosen amino acid.

Example 4 Intracellular Translocation Test of Modified PTD Sequences andeGFP Fusion Proteins in CHO Cells Example 4-1 Construction of eGFPFusion Protein Expression Vector

In evaluating intracellular translocation using eGFP fusion proteins,two kinds of inserts (types C and D in FIG. 14) were prepared.

Primers for insert amplification were designed to allow the in-frameaddition of a His-Tag and a PTD sequence to the N-terminus or C-terminusof eGFP. The design was such that the GGGS (SEQ ID NO: 120) or GGGSS(SEQ ID NO: 121) linker would be encoded in front and back of theHis-Tag and the PTD sequence, respectively. The PTD moiety used was aDNA sequence that encodes PTD1 derived from HIV TAT. Furthermore, theDNA sequence portions of the GGGS (SEQ ID NO: 120) and GGGSS (SEQ ID NO:121) linkers that encode the PTD moiety were prepared to producerecognition sequences for the restriction endonucleases BamHI and XhoI,respectively.

Primer design Insert configuration C (type C in FIG. 14)Forward primer C-F  (SEQ ID NO: 52 in the sequence listing)5′-GCC ATG GTG AGC AAG GGC GAG GAG CTG TTC-3′ Reverse primer C-R1 (SEQ ID NO: 53 in the sequence listing)5′-CAC CGC GGC GAC GTT GTC GTC GTT TCT TCC TGC CGT AGG ATC CCC CTCCCT TGT ACA GCT CGT CCA TGC C-3′ Reverse primer C-R2 (SEQ ID NO: 54 in the sequence listing)5′-CGC TCA GCG TCG ACT CAC CCG TGA TGA TGG TGG TGA TGA CTC GAG CCGCCA CCG CGG CGA CGT TGT CGT-3′Insert configuration D (D type in FIG. 14) Forward primer D-F (SEQ ID NO: 55 in the sequence listing)5′-AAG CCA TGGGAG GGG GATCCT ACG GCA GGA AGA AAC GAC GAC AAC GTCGCC GCG GTG GCG GCT CGA GTA TGG TGA GCA AGG GCG AGG A-3′Reverse primer D-R  (SEQ ID NO: 56 in the sequence listing)5′-CCG CTC AGC GTC GAC TCACCC GTG ATG ATG GTG GTG ATG AGA ACC ACCACC CTT GTA CAG CTC GTC CAT GCC-3′

Thereby, it is possible to cleave the PTD moiety with BamHI and XhoI,and substitute the same with synthetic DNA. With eGFP as the template,the above-described SEQ ID NOS:52 to 56 in the sequence listing wereinserted between the NcoI and Bpu1102I restriction endonucleaserecognition sequences of pET14b (FIG. 15).

The modified PTD sequences were inserted using the synthetic oligosshown in Table 2-1 and Table 2-2, by the method shown in FIG. 16.

TABLE 2-1 forward oligomer SEQ ID NO: in name of modified (F) or reversethe sequence PTD peptide oligomer (R) listing KSH1 F 58 R 59 KSH1-1 F 60R 61 KSH1-2 F 62 R 63 KSH1-3 F 64 R 65 KSH1-4 F 66 R 67 KSH1-5 F 68 R 69KSH1-6 F 70 R 71 KSH1-7 F 72 R 73 KSH1-8 F 74 R 75 KSH1-9 F 76 R 77KSH1-11 F 78 R 79 KSH1-13 F 80 R 81

TABLE 2-2 KSH1-18 F 82 R 83 KSH1-20 F 84 R 85 KSH1-21 F 86 R 87 KSH1-26F 88 R 89 KSH1-27 F 90 R 91 KSH1-28 F 92 R 93 KSH1-30 F 94 R 95 KSH1-33F 96 R 97 KSH1-36 F 98 R 99

Used for negative control was one having His alone added to theC-terminus, amplified using the primer set of SEQ ID NO:52 and 56 in thesequence listing with eGFP as the template.

After the BL21/LysS strain, derived from Escherichia coli strain B, wastransformed with the constructed expression vector, each transformantwas precultured in 20 mL of LB (37° C., 15 hours). The preculture brothwas transplanted to 1 L of LB at 2%, and the transformant was culturedat 37° C. for 2.5 hours. IPTG was added at a final concentration of 1mM, and further cultivation was performed for 4 hours. Aftercentrifugation (4000 rpm, 20 min Hitachi himac CR7) and bacterial cellcollection, the bacterial cells were suspended in 50 mL of PBS.Centrifugation (3500 rpm, 20 min KUBOTA 5200) was performed, andbacterial cells were collected. The bacterial cells were suspended in 30mL of HBSS and freeze-thawed three times. DNase solution (BenzonaseTakara Bio NV677), 15 μL, was added, and the bacterial cells wereincubated at room temperature for 10 minutes. Centrifugation (18000 rpm,20 min TOMY UD-201) was performed, and the supernatant was recovered.The supernatant was applied to an HBSS-equilibrated Ni-NTA column. Afterthe column was washed with 50 mL of an HBSS containing 10 mM imidazole,the fusion proteins were eluted with 4 mL of an HBSS containing 200 mMimidazole.

Example 4-2 Intracellular Translocation of Modified PTD Sequences andeGFP Fusion Proteins in CHO Cells

The intracellular translocation of fusion proteins was investigatedusing CHO cells. CHO cells were inoculated to a 48-well plate at 2×10⁴cells per well; the cells were used after becoming confluent. After thecells were washed with MEM medium three times, 150 μL of proteinsolution was added, and the cells were incubated at 37° C. for 3 hours.After plate washing with PBS three times, 0.25% trypsin/EDTA was addedat 100 μL/well, an MEM medium containing 10% FCS was added at 400μL/well, and the cells were recovered. After being washed with HBSSthree times, the recovered cells were suspended in 100 μL of an HBSScontaining 10% FCS, and subjected to confocal microscopy. The additionconcentration was adjusted to obtain fluorescence intensities of 10⁶,5×10⁶, and 2.5×10⁶ as determined by measuring the amount of eGFP-derivedfluorescence (Ex485 nm/Em535 nm) using ARVO (Perkin-Elmer) (time span ofmeasurement 1 second).

eGFP fusion proteins were prepared with KSH1-1, KSH1-7, KSH1-8, KSH1-9,KSH1-18 and KSH1-33, which exhibited clearly accentuated intracellulartranslocation compared with the KSH1 peptide in Example 3, as well asKSH1-20, KSH1-21, KSH1-26, KSH1-27, KSH1-28, KSH1-30 and KSH1-36. Foranalysis, type C fusion proteins were prepared for KSH1, KSH1-1, KSH1-7and KSH1-8, and type D fusion proteins were prepared for the otherpeptides.

For the prepared eGFP fusion proteins, an intracellular translocationtest using CHO cells was performed; the results are shown in FIG. 17.The upper panels show the fluorescence of eGFP protein that hadtranslocated into the cells; the lower panels show differentialinterference microscopic images of the cells in combination withfluorescence images. The fusion proteins were added to the cells afterthe fluorescence intensities thereof were uniformized to 10⁶. Whenfusion proteins of modified forms of KSH-1 exhibiting intracellulartranslocation and eGFP were added to CHO cells, fluorescence wasdetected in the cells; it was found that all modified forms of KSH-1retained the capability of translocating fusion proteins into cells.Compared with the peptide alone, the fusion proteins showed smallerdifferences in translocation efficiency, but the fusion proteins withKSH1-1, KSH1-7 and KSH1-27 exhibited accentuated intracellulartranslocation compared with KSH1.

As the hydrophobicity of the peptide added increased, the intracellulartranslocation with fusion proteins showed an increased tendency not toreflect the results with the peptide. For KSH1-30, which had twotryptophans substituted with tyrosine, the intracellular translocateddecreased considerably with fusion proteins despite that good resultswere obtained with the peptide; therefore, for substitution of twotryptophans, isoleucine (KSH1-26) was the most suitable.

For replacement of threonine and serine with alanine (KSH1-18, KSH1-20,KSH1-21), no clear accentuation of intracellular translocation wasobserved with fusion proteins. However, when all threonines and serineswere substituted with alanine (KSH1-27), the intracellular translocationtended to be accentuated clearly; therefore, it was concluded that tochange the intracellular translocation efficiency of fusion proteins, alarger structural change in the peptide moiety is required than thepeptide alone.

In summary, with the addition of modified peptides of KSH1, proteinstranslocated into cells, but the translocation efficiency of fusionproteins differed from that of the peptide alone.

Example 5 Intracellular Translocation Test of Novel PTD Sequences (SEQID NOS:39 to 47 in the Sequence Listing) Example 5-1 Selection of NovelPTD Sequences by FACS Analysis

The KSH1 peptide was a sequence obtained from a 7-time-concentratedlibrary of Jurkat cells. Hence, 1000 IVVs contained in the7-time-concentrated library of Jurkat cells were arbitrary picked up,and the DNA sequences thereof were identified. Since analysis of KSH1led to the anticipation that when the peptide sequence was applied tothe wheel structure, the presence of a lysine/arginine cluster andtryptophan would be important, narrowing of 1000 sequences was performedwith these conditions as criteria. As a result, 60 sequences met thecriteria. Of the 1000 sequences, one kind of repeatedly appearingsequence was observed. For these sequences, fluorescently labeledpeptides were synthesized, and their capabilities of translocation intocells were analyzed.

Each peptide was synthesized, fluorescently labeled, purified anddissolved in the same manner as Example 1-2, and an intracellulartranslocation test in CHO cells was performed by FACS analysis. Used forpositive control were a PTD sequence derived from HIV TAT (SEQ ID NO:51in the sequence listing) and the KSH1 peptide; used for negative controlwas a peptide sequence lacking intracellular transferability (SEQ IDNO:57 in the sequence listing).

As a result of the FACS analysis, one kind of sequence exhibiting thesame transferability as KSH1, and nine kinds of sequences exhibiting thesame transferability as TAT-derived PTD, were identified in CHO cells.These nine kinds of peptide sequences are as shown by KSH3 to 10 andKSH2 (SEQ ID NOS:35 to 43 in the sequence listing) (FIG. 18).

Example 5-2 Cellular Transferability Analysis of Novel PTD Sequences byConfocal Microscopy

For KSH2 to KSH10 (SEQ ID NOS:35 to 43 in the sequence listing), a testof translocation into CHO cells was performed in the same manner asExample 1-2, and confocal microscopic analysis was performed. Used forpositive control were a PTD sequence derived from HIV TAT (SEQ ID NO:50in the sequence listing) and the KSH1 peptide; used for negative controlwas a peptide sequence lacking intracellular transferability (SEQ IDNO:57 in the sequence listing).

In the confocal microscopic analysis, a background correction wasperformed using cells to which the negative control peptide (SEQ IDNO:57 in the sequence listing) had been added. Thereafter, the positivecontrol PTD1 peptide (SEQ ID NO:50 in the sequence listing) was added,and fluorescence in the cells was examined.

Regarding novel PTD sequence, each peptide was analyzed in a series of2-fold dilutions at three levels from 50 μM to 12.5 μM. As a result, theresults of the confocal microscopic analysis showed the same tendency asthe results of the FACS analysis. For the four kinds of KSH2, KSH3,KSH4, KSH6, KSH7 and KSH10, the results of the confocal microscopicanalysis, like the FACS analysis, revealed better intracellulartranslocation profiles than PTD1 (FIG. 19, the upper panels show thefluorescence of fluorescence-labeled peptides translocated into cells;the lower panels show differential interference microscopic images ofcells in combination with fluorescence images; the peptide additionconcentration was 25 μM). The PTD candidate sequences identified hereclearly retained the capability of intracellular translocation. Whendatabase search was performed, no homologous sequences were observed;all were found to be novel PTD sequences. Regarding KSH2, the peptidewas localized as fringing the surface of the cell membrane in HeLacells, showing a transferability pattern different from that of otherPTDs.

Example 5-3 Intracellular Translocation Test of Novel PTD Sequences andeGFP Fusion Proteins in CHO Cells

For the six kinds of KSH2, KSH3, KSH4, KSH6, KSH7 and KSH10, eGFP fusionproteins were prepared, and whether the sequences had a PTD-likeactivity to translocation novel PTD sequences and eGFP fusion proteinsinto cells was determined. For the fusion proteins, molecular forms ofhigh solubilization state were chosen. As a result, for KSH2 and KSH6,molecular type C in FIG. 14 was chosen, and for the remaining fourkinds, type D in FIG. 14 was chosen. The fusion proteins were preparedin the same manner as Example 4-1. The annealing oligo-sequences usedhere are as shown in Table 3. Used for negative control was eGFP-His;after respective fluorescence intensities were uniformized, therespective fusion proteins were added to the CHO cells.

forward oligomer SEQ ID NO: in name of modified (F) or reverse thesequence PTD peptide oligomer (R) listing KSH3 F 100 R 101 KSH4 F 102 R103 KSH6 F 104 R 105 KSH7 F 106 R 107 KSH10 F 108 R 109 KSH2 F 110 R 111After incubation at 37° C. for 1 hour, trypsinization was performed, andthe cells were recovered. After washing operation, the presence orabsence of translocation into cells was determined using a confocalmicroscope. In performing a measurement, conditions were establishedunder which fluorescence was not detected in CHO cells to which negativecontrol eGFP-His had been added, after which measurements of cells towhich each novel PTD fusion eGFP protein had been added were performed.As a result, fluorescence was observed in CHO cells to which anyprepared novel PTD fusion eGFP protein had been added, demonstrating thetranslocation of the fusion proteins into cells.

From this finding, it was found that the above-described six kinds ofnovel PTD sequences had PTD-like activity. For the KSH7 peptide, basicamino acids shared by known PTDs that have been reported to date, suchas arginine, were present alone, representing a new category of PTD. Ofthe six kinds of novel PTDs, KSH4 and KSH2 exhibited highertranslocation into cells than the other novel PTDs and KSH1 peptide, andwere useful when a protein, particularly eGFP, was used as the cargo.

Example 6 Intracellular Translocation Tests of Modified PTD SequencePeptides of SEQ ID NO:47 in CHO Cells Example 6-1 IntracellularTranslocation Test of Modified PTD Sequence Peptides Using a ConfocalMicroscope

Of the novel PTD sequences found in Example 5, the KSH2 peptide ishighly capable of translocating eGFP into cells; an attempt was made tomodify the KSH2 peptide. The KSH2-1 to KSH2-4 peptides (SEQ ID NOS:44 to47 in the sequence listing) were synthesized, fluorescently labeled,purified and dissolved in the same manner as Example 1-2, a test oftranslocation into CHO cells was performed, and confocal microscopicanalysis was performed.

The KSH2 peptide is characterized by the formation of a lysine-argininecluster on the wheel structure and the presence of two tryptophans.Another characteristic is that two cysteines are present as sandwichingtryptophan. From analyses that have been performed to date, it has beenfound that a basic amino acid cluster and tryptophan are important.Hence, in modifying the KSH2 peptide, the influence of substitution ofthe two cysteines with other amino acids was examined (FIG. 20).

First, two cases were examined: two cysteines were substituted withalanine (KSH2-1) or with tryptophan (KSH2-2). FITC-labeled peptides weresynthesized, and their intracellular translocation efficiencies wereexamined using CHO, HeLa, and Jurkat cells (FIG. 21, the peptideaddition concentration was 50 μM, 25 μM or 12.5 μM).

As a result, at a concentration of 50 μM, both KSH2-1 and KSH2-2exhibited higher intracellular translocation than KSH2; when diluted to25 μM, KSH2-1 exhibited remarkably decreased intracellulartranslocation. Meanwhile, KSH2-2 exhibited higher intracellulartranslocation efficiency than KSH2 even when diluted to 25 μM and 12.5μM. In KSH2-2, which exhibited promoted intracellular translocation, thenumber of tryptophans was 4. As the number of tryptophans increases, theintracellular translocation of a peptide is accentuated; however, sincethis was thought to be likely to cause insolubilization in preparing afusion protein, two cysteines were substituted one by one with alanine(KSH2-3 and KSH2-4), and the intracellular translocation was examined.When the capability of intracellular translocation of FITC-labeledpeptides in CHO, HeLa, and Jurkat cells was examined, it was found thatin both cases, the capability of intracellular translocation wasaccentuated, compared with KSH2.

Example 6-2 Intracellular Translocation Test of Fusion Proteins ofModified PTD Sequences and eGFP in CHO Cells

For KSH2-3 and KSH2-4 (SEQ ID NOS:46 and 47 in the sequence listing),eGFP fusion proteins were prepared, and the transferabilities of KSH2and eGFP fusion proteins were compared in terms of PTD-like activity totranslocation the novel PTD sequences and eGFP fusion proteins intocells. The fusion proteins prepared were type C in FIG. 14 for KSH2 andtype D in FIG. 14 for KSH2-3 and KSH2-4. The fusion proteins wereprepared in the same manner as Example 4-1. The annealingoligo-sequences used here are as shown in Table 4.

forward oligomer SEQ ID NO: in name of modified (F) or reverse thesequence PTD peptide oligomer (R) listing KSH2-3 F 112 R 113 KSH2-4 F114 R 115When the capability of translocation into CHO cells was examined,translocation into cells was confirmed for both fusion proteins ofKSH2-3 and KSH2-4 with eGFP. Regarding intracellular translocationefficiency, the capability of translocation was lower than that of theeGFP fusion protein with the KSH2 peptide, but was equivalent to that ofTAT-derived PTD1.

In summary, it was concluded that it is efficient to choose KSH2 when apolymeric protein is used as the cargo, and to choose the modified formsKSH2-3 and KSH2-4 when low-molecular substances like peptides are used.

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to provide a cellpenetrating peptide having a novel amino acid sequence and apharmaceutical containing the peptide. This application claims prioritybased on a Japanese patent application No. 2005-314355.

1.-4. (canceled)
 5. An isolated polynucleotide that encodes (i) a peptide having an amino acid sequence: B-X-Z-X-Arg-Z-Tyr-J-X-O¹-X-Arg-O²-X-X or X-X-O¹-Arg-X-O²-X-J-Tyr-Z-Arg-X-Z-X-B;

wherein (i) B is arginine or lysine, (ii) at least one of O¹ or O² is arginine, (iii) Z is a hydrophobic amino acid, (iv) J is serine or alanine, and (v) X is an arbitrarily chosen amino acid; (ii) a peptide consisting of the amino acid sequence shown by any one of SEQ ID NOS:1 to 34 in the sequence listing wherein one or a plurality of amino acids have been substituted, deleted, added or inserted; (iii) a peptide consisting of the amino acid sequence shown by any one of SEQ ID NOS:35 to 47 in the sequence listing wherein one or a plurality of amino acids have been substituted, deleted, added or inserted, and the reversed-chain peptide thereof; or (iv) a peptide consisting of the amino acid sequence shown by any one of SEQ ID. NOS:1 to 47 in the sequence listing, and the reversed-chain peptide thereof.
 6. A recombinant vector comprising the isolated polynucleotide of claim
 5. 7. A transformant comprising the recombinant vector of claim
 6. 8. A method for making a peptide comprising cultivating the transformant of claim 7 and recovering said peptide; wherein said peptide is selected from the group consisting of (i) a peptide having an amino acid sequence: B-X-Z-X-Arg-Z-Tyr-J-X-O¹-X-Arg-O²-X-X or X-X-O¹-Arg-X-O²-X-J-Tyr-Z-Arg-X-Z-X-B;

wherein (i) B is arginine or lysine, (ii) at least one of O¹ or O² is arginine, (iii) Z is a hydrophobic amino acid, (iv) J is serine or alanine, and (v) X is an arbitrarily chosen amino acid; (ii) a peptide consisting of the amino acid sequence shown by any one of SEQ ID NOS:1 to 34 in the sequence listing wherein one or a plurality of amino acids have been substituted, deleted, added or inserted; (iii) a peptide consisting of the amino acid sequence shown by any one of SEQ ID NOS:35 to 47 in the sequence listing wherein one or a plurality of amino acids have been substituted, deleted, added or inserted, and the reversed-chain peptide thereof; and (iv) a peptide consisting of the amino acid sequence shown by any one of SEQ ID NOS:1 to 47 in the sequence listing, or the reversed-chain peptide thereof.
 9. The isolated polynucleotide of claim 5 that encodes (i) a peptide having the amino acid sequence: B-X-Z-X-Arg-Z-Tyr-J-X-O¹-X-Arg-O²-X-X or X-X-O¹-Arg-X-O²-X-J-Tyr-Z-Arg-X-Z-X-B; wherein (i) B is arginine or lysine, (ii) at least one of O¹ or O² is arginine, (iii) Z is a hydrophobic amino acid, (iv) J is serine or alanine, and (v) X is an arbitrarily chosen amino acid.
 10. The isolated polynucleotide of claim 5 that encodes (ii) a peptide consisting of the amino acid sequence shown by any one of SEQ ID NOS:1 to 34 in the sequence listing wherein one or a plurality of amino acids have been substituted, deleted, added or inserted.
 11. The isolated polynucleotide of claim 5 that encodes (iii) a peptide consisting of the amino acid sequence shown by any one of SEQ ID NOS:35 to 47 in the sequence listing wherein one or a plurality of amino acids have been substituted, deleted, added or inserted, and the reversed-chain peptide thereof.
 12. The isolated polynucleotide of claim 5 that encodes (iv) a peptide consisting of the amino acid sequence shown by any one of SEQ ID NOS:1 to 47 in the sequence listing, and the reversed-chain peptide thereof. 13.-15. (canceled)
 16. An isolated polynucleotide encoding a peptide having the capability of translocation to cells, wherein said peptide is one of the following (a) or (b): (a) a peptide comprising the amino acid sequence of any one of SEQ ID NOS: 3-5, 14, 16, 21, 31 and 35-47 or the reversed-chain peptide thereof; (b) a peptide comprising the amino acid sequence of (a) above wherein one or two amino acids have been substituted, deleted, added or inserted; or the reversed chain peptide thereof.
 17. The isolated polynucleotide of claim 16 that is (a) a peptide comprising the amino acid sequence of any one of SEQ ID NOS: 3-5, 14, 16, 21, 31 and 35-47 or the reversed-chain peptide thereof.
 18. The isolated polynucleotide of claim 16 that is (b) a peptide comprising the amino acid sequence of (a) above wherein one or two amino acids have been substituted, deleted, added or inserted or the reversed chain peptide thereof.
 19. A recombinant vector comprising the isolated polynucleotide of claim
 16. 20. A transformant comprising the recombinant vector of claim
 19. 21. A method for making a peptide comprising cultivating the transformant of claim 20 and recovering said peptide; wherein said peptide is one of the following (a) or (b): (a) a peptide comprising the amino acid sequence of any one of SEQ ID NOS: 3-5, 14, 16, 21, 31 and 35-47 or the reversed-chain peptide thereof; (b) a peptide comprising the amino acid sequence of (a) above wherein one or two amino acids have been substituted, deleted, added or inserted or the reversed chain peptide thereof. 